IntAct European Bioinformatics Institute IntAct intact http://www.ebi.ac.uk/ http://www.ebi.ac.uk/intact/query/${ac} EBI-[0-9]+|IA:[0-9]+ INTerAction database (IntAct) provides an open source database and toolkit for the storage, presentation and analysis of molecular interactions. http://www.ebi.ac.uk/intact European Bioinformatics Institute; Wellcome Trust Genome Campus; Hinxton, Cambridge; CB10 1SD; United Kingdom http://www.ebi.ac.uk/intact/ Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. J. Biol. Chem. (0021-9258) 2007 imex curation Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis mw2025@columbia.edu 27-Aug-2008: Contacted by JYOTI. The mouse brain cDNA library pPC86 (Invitrogen) was used. davet Only protein-protein interactions human-293 Homo sapiens transformed primary embryonal kidney cells Human 293 Transformed human primary embryonal kidney cells. The Cell type named 293 is synonymous with HEK-293. anti tag coip anti tag coimmunoprecipitation anti tag coip anti tag western anti tag western blot anti tag western Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis The range for the Sh3rf1 (POSH) Zinc finger domain was taken from PMID 9482736. mw2025@columbia.edu Accepted 2008-JUN-23 by DAVET. 27-Aug-2008: Contacted by JYOTI. The mouse brain cDNA library pPC86 (Invitrogen) was used. J. Biol. Chem. (0021-9258) 2007 davet Only protein-protein interactions imex curation Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. J. Biol. Chem. (0021-9258) 2007 imex curation Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis mw2025@columbia.edu 27-Aug-2008: Contacted by JYOTI. The mouse brain cDNA library pPC86 (Invitrogen) was used. davet Only protein-protein interactions yeasx Saccharomyces cerevisiae (Baker's yeast) Baker's yeast 2 hybrid two hybrid 2H 2h Gal4 transcription regeneration two-hybrid yeast two hybrid 2-hybrid classical two hybrid classical two hybrid 2H two-hybrid 2-hybrid Gal4 transcription regeneration 2h yeast two hybrid Y2H Y-2H 2 hybrid nucleotide sequence nucleotide sequence identification sequence cloning sequence cloning nucleotide sequence Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis mw2025@columbia.edu Accepted 2008-JUN-23 by DAVET. The mouse brain cDNA library pPC86 (Invitrogen) was used. J. Biol. Chem. (0021-9258) 2007 davet Only protein-protein interactions imex curation Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. Proapoptotic Nix activates the JNK pathway by interacting with POSH and mediates death in a Parkinson disease model. J. Biol. Chem. (0021-9258) 2007 imex curation Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis mw2025@columbia.edu 27-Aug-2008: Contacted by JYOTI. The mouse brain cDNA library pPC86 (Invitrogen) was used. davet Only protein-protein interactions in vitro In vitro pull down pull down weight autoradiogra molecular weight estimation by autoradiography weight autoradiogra Wilhelm M., Xu Z., Kukekov NV., Gire S., Greene LA. Apoptosis - Interactions involving proteins with a function related to apoptosis mw2025@columbia.edu Accepted 2008-JUN-23 by DAVET. 27-Aug-2008: Contacted by JYOTI. The mouse brain cDNA library pPC86 (Invitrogen) was used. J. Biol. Chem. (0021-9258) 2007 davet Only protein-protein interactions imex curation bni3l_mouse BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like Bnip3l Nix NIP3-like protein X protein protein mouse Mus musculus Mouse MSHLVEPPPPLHNNNNNCEEGEQPLPPPAGLNSSWVELPMNSSNGNENGNGKNGGLEHVPSSSSIHNGDMEKILLDAQHESGQSSSRGSSHCDSPSPQEDGQIMFDVEMHTSRDHSSQSEEEVVEGEKEVEALKKSADWVSDWSSRPENIPPKEFHFRHPKRAASLSMRKSGAMKKGGIFSAEFLKVFIPSLFLSHVLALGLGIYIGKRLSTPSASTY EAA839DEFDDE50D7 sh3r1_mouse E3 ubiquitin-protein ligase SH3RF1 Posh1 Sh3rf1 Kiaa1494 Posh SH3 domain-containing RING finger protein 1 Plenty of SH3s SH3 multiple domains protein 2 Sh3md2 RING-type E3 ubiquitin transferase SH3RF1 protein protein mouse Mus musculus Mouse MDESALLDLLECPVCLERLDASAKVLPCQHTFCKRCLLGIVGSRNELRCPECRTLVGSGVDELPSNILLVRLLDGIKQRPWKPGPGGGGGTTCTNTLRAQGSTVVNCGSKDLQSSQCGQQPRVQAWSPPVRGIPQLPCAKALYNYEGKEPGDLKFSKGDIIILRRQVDENWYHGEVSGVHGFFPTNFVQIIKPLPQPPPQCKALYDFEVKDKEADKDCLPFAKDDVLTVIRRVDENWAEGMLADKIGIFPISYVEFNSAAKQLIEWDKPPVPGVDTAECPSATAQSTSASKHPDTKKNTRKRHSFTSLTMANKSSQGSQNRHSMEISPPVLISSSNPTAAARISELSGLSCSAPSQVHISTTGLIVTPPPSSPVTTGPAFTFPSDVPYQAALGSMNPPLPPPPLLAATVLASTPSGATAAVAAAAAAAAAAGMGPRPVMGSSEQIAHLRPQTRPSVYVAIYPYTPRKEDELELRKGEMFLVFERCQDGWYKGTSMHTSKIGVFPGNYVAPVTRAVTNASQAKVSMSTAGQASRGVTMVSPSTAGGPTQKPQGNGVAGNPSVVPTAVVSAAHIQTSPQAKVLLHMSGQMTVNQARNAVRTVAAHSQERPTAAVTPIQVQNAACLGPASVGLPHHSLASQPLPPMAGPAAHGAAVSISRTNAPMACAAGASLASPNMTSAMLETEPSGRTVTILPGLPTSPESAASACGNSSAGKPDKDSKKEKKGLLKLLSGASTKRKPRVSPPASPTLDVELGAGEAPLQGAVGPELPLGGSHGRVGSCPTDGDGPVAAGTAALAQDAFHRKTSSLDSAVPIAPPPRQACSSLGPVMNEARPVVCERHRVVVSYPPQSEAELELKEGDIVFVHKKREDGWFKGTLQRNGKTGLFPGSFVENI D92071116F4D6EBE sh3rf1-bnip3l 2 5 unspecified role unspecified role bait bait 4 unspecified role unspecified role prey prey physical association physical association To identify additional POSH-binding partners, we performed a yeast twohybrid analysis using POSH cloned into a bait vector as previously described (18). One clone corresponded to Nix, a proapoptotic BH3-only protein sh3rf1-bnip3l-1 1 4 unspecified role unspecified role prey prey region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined 5 unspecified role unspecified role bait bait region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range certain certain sequence position certain certain certain sequence position certain range range physical association physical association 1 A FLAG-Nix was detected only in immunoprecipitates from cells expressing Myc-ZnPOSH The Sh3rf1 (POSH) construct had the Zinc finger domain deleted to enhance protein stability while retaining the ability to activate the JNK pathway. O70254; Q9Z2F7 transfection As the coordinates for the Zn finger domain deleted from Sh3rf1 (POSH) are taken from a paper by different authors, these are given as fuzzy coordinates. sh3rf1-bnip3l-2 1 5 unspecified role unspecified role prey prey region myc tag myc tag EUKLISEED epitope tag EUKLISEED epitope tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined region sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis range range certain certain sequence position certain certain certain sequence position certain range range 4 unspecified role unspecified role bait bait region flag tag flag tag FLAG-tagged FLAG DYKDDDDKV epitope tag DYKDDDDKV epitope tag FLAG FLAG-tagged undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined physical association physical association 1 B The Sh3rf1 (POSH) construct had the Zinc finger domain deleted to enhance protein stability while retaining the ability to activate the JNK pathway. O70254; Q9Z2F7 transfection we also performed reciprocal immunoprecipitations using anti-FLAG. Fig. 1B shows that Nix and ZnPOSH again specifically co-immunoprecipitated under these conditions. As the coordinates for the Zn finger domain deleted from Sh3rf1 (POSH) are taken from a paper by different authors, these are given as fuzzy coordinates. sh3rf1-bnip3l-3 3 4 unspecified role unspecified role prey prey region 35s radiolabel 35s radiolabel 35S s35 radiolabelled S35 s35 radiolabelled 35S S35 undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined n-terminal sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain region DeltaBH3 sufficient to bind sufficient binding region sufficient to bind deletion analysis deletion analysis certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain certain certain sequence position certain in vitro In vitro 5 unspecified role unspecified role bait bait region gst tag glutathione s tranferase tag glutathione S-tranferase tag glutathione S-tranferase tag gst tag undetermined undetermined sequence position undetermined undetermined undetermined sequence position undetermined ecoli Escherichia coli (strain K12) physical association physical association 1 C Nix binds directly to POSH Sh3rf1 (POSH) also bound to a naturally occurring dominant-negative isoform of Bnip3l (Nix), sNix, (which lacks only the terminal 10 amino acids of the protein and unlike Bnip3l, is not targeted to mitochondrial membranes) and Bnip3l lacking the BH3 domain.